WO2012175454A2

WO2012175454A2 - In vitro assay method using immunological technique

Info

Publication number: WO2012175454A2
Application number: PCT/EP2012/061603
Authority: WO
Inventors: Sébastien Bonnet; Delphine Rival
Original assignee: Basf Beauty Care Solutions France S.A.S.
Priority date: 2011-06-20
Filing date: 2012-06-18
Publication date: 2012-12-27
Also published as: EP2721408B1; US20140162284A1; US20130017537A1; FR2976587B1; CN103688170A; WO2012175454A3; EP2721408A2; US9146237B2; FR2976587A1; ES2656952T3

Abstract

The present invention relates to a novel method for the in vitro assay of molecules from the extracellular matrix that are synthesized by cultured cells, and to the uses thereof as an in vitro measuring kit and/or as a method for screening for cosmetic and/pharmaceutical ingredients.

Description

The present invention relates to a novel method for in vitro assay of extracellular matrix molecules synthesized by cells in culture and its uses in the form of in vitro measurement kit and / or screening method of cosmetic and / or pharmaceutical ingredients.

The extracellular matrix (ECM) plays an essential role in the structure of the tissues of the human and animal body, in particular by its functions of support, adhesion and regulation of cellular exchanges. ECM consists largely of glycoproteins, proteins and glycosaminoglycans. These molecules are synthesized in native form in the cells in contact with the MEK. From the intracellular compartment, they are excreted outside the cells. After maturing phenomena, they organize and combine in a network that forms the MEC. They are then in their functional form. The molecules of the MEC are particularly studied in the cosmetic and pharmaceutical fields where many ingredients are designed to stimulate

Their synthesis and thus improve the general state of the tissue concerned.

In vitro, the cells in culture under appropriate conditions produce their extracellular matrix and constitute a particularly simple model of studies, in particular to measure the synthesis activity by the cells of one or more molecules constituting the ECM and the influence of ingredients on this

20 activity. In this type of cell model in culture, the molecules of the ECM are present in different culture compartments and optionally in different forms including precursors: intracellular, in the culture medium, or contained in the ECM.

Conventional measurement techniques involve measuring the amount of

25 molecules of ECM in the culture medium. Thus, according to the most common technique, ECM molecules such as collagens, optionally in their precursor form, such as procolagen, are assayed in the culture medium by ELISA technique. Another more expensive, more restrictive and more comprehensive technique is to include in the culture medium

30 radiolabeled molecules used by cells for the synthesis of molecules of the ECM and to measure overall the intensity of the radioactivity thus obtained in all the compartments. For example, tritiated proline is used to measure the amount of collagen globally produced by cells in culture. These techniques make it possible to account for the activity of synthesis of the cells in culture but not for the functionality of the molecules thus synthesized, that is to say of the quantity of molecules in the MEK.

Only immunohistochemical analysis can determine and only partly the functionality of the ECM molecules thus synthesized. This technique consists in eliminating the culture medium, labeling with antibodies directed against the ECM molecules studied, and then reading and / or visualizing the fluorescence. This technique makes it possible to quantify the molecules contained in the intracellular, extracellular compartment and in the ECM and especially to visualize their location. This technique is nevertheless cumbersome to implement and requires expensive equipment. The technique itself does not

In addition, it does not allow the analysis of a large number of ingredients. Moreover, it is subjective and depends on the experimenter. Thus, prior to the present invention, no objective quantitative measurement made it possible to determine the quantity of functional molecules that is to say the molecules actually integrated in the MEK and in particular the molecules that constitute the MEK in a manner that

20 sufficiently fast, reliable, simple and predictive. Moreover, no method allowed the screening of ingredients of cosmetic and / or pharmaceutical interest.

However, an important synthesis of ECM molecules by cultured cells measured in the intracellular and / or extracellular medium does not necessarily result in a larger quantity of functional molecules, ie molecules in the MEK. .

Indeed, the Applicant has found that certain cosmetic active ingredients can globally stimulate the collagen synthesis of the ECM without, however, visualizing an improvement in the functionality of the collagen, that is to say, no increase in the content. collagen in the MEC. The Applicant has also found that certain ingredients improve the functionality of the collagen that is to say its content in the ECM without, however, measuring an increase in procollagen synthesis. (examples 6 and 7)

The techniques of the prior art are therefore not appropriate for quantifying the functionality of the ECM molecules, ie their content in the ECM in that they are not direct or reliable enough, predictive, reproducible, repeatable. and specific. Furthermore, they do not allow the screening or "screening" of cosmetic and / or pharmaceutical ingredients that can improve the functionality of the MEK molecules in a satisfactory manner and to visualize the effects thereof in the MEK.

Diquelou A. et al., 1995, Relationship between endothelial tissue factor and thrombogenesis under blood conditions, Thrombosis and haemostasis, vol 74, No. 2, p778-783 and Ryan J. et al, 1992, Tumor necrosis factor-induced endothelial tissue factor is associated with subendothelial matrix vesicles but is

Noted on the Apical Surface, Blood, Vol 80, No. 4, p966-974 described the assay of HUVECS synthesized TF after 4 or 8 h of culture. Nevertheless, such culture times are insufficient for cultured cells to synthesize an ECM and to study the functionality of the constitutive and / or contained molecules in the ECM. These experiences have moreover

20 different purpose.

There was therefore a need to have a technique for assaying MEC molecules synthesized by cultured cells which makes it possible to directly, quantitatively, reliably, simply, repeatably, predictively and specifically assay the functionality of the MEC molecules. that is to say

25 their content in the MEK. There was also a need for a non-denaturing MEK assay technique that quantifies and visualizes the molecule studied in the MEK and to study it in situ, particularly in its functional form and in its interactions with other molecules of the ECM. The Applicant has now surprisingly and unexpectedly discovered that this problem can be solved by performing a step of specific lysis of the cells in culture and a direct assay on the ECM of the molecules of interest. The method according to the invention thus provides a technical solution to the problem of the prior art and has the advantage of being rapid, miniaturizable, and of allowing the screening of a large number of ingredients for their capacity to increase or to reduce the quantity of molecules in the ECM, advantageously in automatable form. The method according to the invention also has the advantage of not destroying the MEK and of allowing both quantification and visualization of the MEK molecules.

The subject of the present invention is a method for in vitro assay by immunological technique of at least one molecule of the extracellular matrix synthesized by cells in culture comprising at least the steps:

a) a step of culturing the cells and preferentially culturing the confluent cells for at least 24 hours,

b) a preferential step of sampling the culture medium,

c) a step of lysing the cells with a quaternary amine solution, preferably with a quaternary amine solution at a concentration of between 100 μΜ and 2M,

D) a cell lysate removal step ,.

e) a step of immunologically assaying said molecule in the extracellular matrix.

The present invention further relates to a method for in vitro assay by immunological technique of at least one extracellular matrix molecule synthesized by cells in culture comprising at least the steps:

b) a step of collecting the culture medium and assaying the ECM molecule and / or a precursor of the ECM molecule, c) a step of lysing the cells with a quaternary amine solution, preferably with a quaternary amine solution at a concentration of between 100 μΜ and 2M,

d) a step of collecting the cell lysate and assaying the DNA and / or assaying the ECM molecule and / or a precursor of the ECM molecule, e) a step of assaying by immunological technique of said molecule of the ECM contained in the extracellular matrix.

The present invention also relates to the use of the assay method according to the invention for the screening and / or the in vitro study of at least one ingredient of cosmetic and / or pharmaceutical interest for its properties. increase or decrease the amount of a molecule in the extracellular matrix.

The present invention also relates to a method for screening and / or in vitro study of at least one ingredient of cosmetic and / or pharmaceutical interest for its properties to increase or decrease the amount of a molecule in the matrix. extracellular composition comprising at least the steps:

a) a step of culturing the cells and culturing the cells in the presence of the ingredient, preferably for at least 24 hours after confluence, b) preferentially a step of taking the culture medium and, still more preferably, assaying the molecule of the CME and / or a precursor of the

20 molecule of the ECM in said culture medium thus collected,

c) a step of lysing the cells with a quaternary amine solution, preferably with a quaternary amine solution at a concentration of between 100 μΜ and 2 M

d) a step of sampling the cell lysate and preferably of assaying the DNA and / or assaying the ECM molecule and / or a precursor of the ECM molecule,

e) a step of immunological assay of said molecule of the ECM contained in the extracellular matrix. Another subject of the present invention is an immunoassay kit useful according to one of the methods according to the invention comprising at least one quaternary amine solution, preferably a quaternary amine solution at a concentration of between 100 μΜ and 2M, and an antibody directed against the ECM molecule, optionally coupled to a secondary antibody:

Another subject of the present invention is a method for selecting a lysis solution that is useful in the assay method according to the invention, characterized in that the assay method according to the invention is implemented with a solution of lysis test for at least 10 min, preferably 10 min, and the ratio of the result of the MEK molecule assay obtained in step e) on the result of the assay of the DNA in the cell lysate is compared to that obtained with a lysis solution of ammonium hydroxide at 20 mM applied during the same time, preferably 10 minutes.

According to the invention, the term "ECM molecules" means the organic molecules which constitute and / or are contained in the extracellular matrix and which are synthesized by cells, of human or animal origin.

These include the constitutive proteins of the ECM, in particular selected from:

The collagen family of the ECM: fibrillar collagens, in particular of type I, I II, V, FACITS (Fibril Associated Collagen with I nterupted Tri ple Helix) collagens, especially of type VI, XI I, XIV, XVI, the collagens IV and VII, collagens XXII, XXVII, XVIII

- The family of elastic fibers of the ECM: elastin, tropoelastin,

25 proteins associated with elastin including fibrillin 1, lysyl oxidases, in particular LOX and LOXL, ΙΈΒΡ (Elastin Binding Protein), fibullins 3 and 5, Emilin 1 and 2.

These include the proteoglycans constituting the ECM, in particular the secreted proteoglycans chosen from Perlecan, Versican, leucine-rich proteoglycans (SLRPs) including decorin, biglycan and lumican.

They are also constituent glycoproteins of the MEK, in particular fibronectin, laminin, SPARC (Secreted Protein Rich in Cystein), tenascine, nidogen 1.

It is also glycosaminoglycans (GAG) constitutive of ECM, in particular hyaluronic acid, heparan sulfate, dermatan sulfate, chondroitin sulfate.

It is also the growth factors of the ECM which are proteins contained in the MEK, in particular VEGF, PDGF, HGF, FGF, and in particular FGF2 and FGF7.

The present invention very advantageously makes it possible to assay the constitutive molecules of the MEK and to visualize them in situ in the well, that is to say without destroying the MEK.

Some ECM molecules exist in other compartments than the MEK as a precursor.

The "molecules of the ECM" denote the molecules in the MEK or in other compartments, in particular intracellular and extracellular compartments in the culture medium, possibly in the form of precursors.

According to the invention, the term "MEC molecule precursor" means a native and / or intermediate form of the MEC molecule, synthesized by the cell, before constituting or being contained in the MEC. These forms undergo maturation phenomena before being in the ECM and are thus often designated by the prefixes "Pro-" or "Tropo-". These are for example the

Procollagen, tropocollagen, proelastine, tropoelastine.

According to the invention, the term "cell (s) in culture" means human or animal cells capable of synthesizing one or more molecules of the ECM. These cells in culture are cultured according to the method according to the present invention and the ECM molecule or molecules that they synthesized are assayed. according to the present invention. They are preferably of differentiated type, especially in the form of differentiated cell precursors or differentiated non-precursor cells. They may be normal, mutated or immortalized, grown in monolayers, possibly in co-culture with one or more other cell types capable or not of synthesizing one or more molecules of the ECM. These cells are notably stromal cells, preferentially fibroblasts, osteoblasts and / or adipoblasts, preadipocytes, adipocytes, epithelial cells, preferentially keratinocytes or endothelial cells. They are extracted from biopsies, preferentially skin, or cell lines. According to a preferred embodiment, the present invention is particularly advantageous for the study of constitutive molecules of ECM synthesized by fibroblasts in culture. Indeed, a functional ECM is essential in the structure of the cutaneous tissues and the method according to the present invention

This access is possible in the culture well, including after 2 to 4 days of postconfluence fibroblast culture.

According to the invention, the term "immunological method" is used to mean quantitative antibody determination techniques directed against proteins or sugars.

These are EIA enzyme-substrate assay techniques (Enzyme

Immuno Assay), including ELISA (Enzyme-Linked Immunosorbent Assay), radiolabelling said RIA (radioimmunoassay) and fluorescence measurement said FIA (Fluorescent immunoassay) especially in time-delayed fluorescence called TRF (Time Resolved fluorescence).

According to the invention, the term "ingredient of cosmetic and / or pharmaceutical interest", one or more natural molecules and / or syntheses, and / or a plant extract, optionally synthesized and / or modified by biological engineering, in particular by microorganism fermentation, whose properties on the molecules of the ECM are evaluated for its ability to

To increase or decrease the amount of molecule in the MEK. The object according to the invention is a method for in vitro assay by immunological technique of at least one molecule of the extracellular matrix synthesized by cells in culture comprising at least the successive steps:

step of culturing the cells

a preferred step of sampling the culture medium

step of lysis of the cells with a quaternary amine solution

- Cell lysate removal step

- Assaying step by immunological technique of said molecule contained in the extracellular matrix.

The method according to the invention makes it possible to assay at least one molecule of the

MEC and advantageously one or two, more preferably at least two.

According to a preferred embodiment, the ECM molecules are chosen from the constitutive proteins of the ECM, the constitutive glycoproteins of the ECM, the constituent glycoaminoglycans of the ECM, the proteoglycans constituting the ECM and the growth factors contained in the ECM. More preferably, they are chosen from collagens of type I, III, V, VI, XII, XIV, XVI, IV and VII, elastin, tropoelastin, fibrillin 1, LOX, LOXL, ΙΈΒΡ (Elastin Binding Protein). , fibullins 3 and 5, Emilin 1 and 2, Perlecan, versican, decorin, biglycan, lumican, fibronectin, laminin, hyaluronic acid, heparan sulfate, VEGF, PDGF, FGF -2, FGF-7, and HGF.

According to a particularly advantageous mode, the molecules of the ECM are selected from collagens type I, I, I, V, XVIII, IV, VII, perlecan, fibronectin, FGF-2, elastin, hyaluronic acid. In a preferred embodiment, the ECM molecules are selected from type I, III, V, XVIII, IV and VII collagens.

The cells capable of synthesizing the ECM molecules are cultured in vitro preferably in monolayer according to the usual techniques in the field. They are sown on a growing medium suitable, preferably a cell culture plate comprising wells, and cultured in a suitable culture medium up to the confluence stage, preferably between 80 and 100% confluence, more preferably between 90 and 100%, more preferably 100%.

5

For the use of the medium flow screening method, the plates are advantageously 96 or 384 well plates. Conventionally the plates can be "coated" that is to say treated to improve the adhesion of cells by a coating which may be in particular constitutive molecules of the ECM. In a preferred manner, the plates will be uncoated or coated with a molecule different from the one whose measurement is carried out and / or whose content is studied in the MEK. According to a preferred embodiment, the cells in culture are chosen from fibroblasts, preferentially dermal and / or keratinocytes and / or adipocytes, advantageously human,

And preferably extracted from biopsies, preferentially from skin, preferably normal human cells, that is to say non-immortalized non-mutated cells. The studies carried out have indeed shown that the method according to the invention is particularly suitable for these cell types.

It is generally at the confluence stage that the cells will begin to secrete the ECM molecules. Advantageously, the cells are maintained in culture at the confluent stage for a period of at least 24 hours and preferably up to 6 days, preferably ranging from 24 to 96 hours, and even more preferably for 48 hours. In a particularly advantageous mode, the collagen is the molecule of the MEC assayed and the fibroblast culture is advantageously maintained for about 48 hours.

According to one embodiment, at confluence, an ingredient of cosmetic and / or pharmaceutical interest is added to the culture medium. It may also be a control ingredient for stimulation or inhibition of ECM molecules. Preferably, the stimulation control is chosen from preferentially vitamin C and beta TGF, and advantageously when the cells in culture are fibroblasts. Vitamin C is then advantageously used at a dose in a range from 5μΜ to 100μΜ, more preferably 50μΜ. The beta TGF is then advantageously used at a dose ranging from 1 ng / ml to 100 ng / ml, preferentially at 10 ng / ml.

Preferably, the culture of confluent cells in the culture medium containing the ingredient of cosmetic and / or pharmaceutical interest is carried out for at least 24 hours, preferably up to 6 days and preferably from 24 to 96 hours. , preferably for 48 hours.

A step of sampling the total or partial culture medium is then preferably carried out. According to an advantageous embodiment, the molecule of the ECM studied and contained in the culture medium, optionally in a precursor form, is assayed by immunological method, preferably by ELISA type assay.

The method according to the invention then comprises a step of lysis of the cells in culture with a quaternary amine solution, preferably an aqueous solution, more preferably an ammonium solution optionally in the form of salts. In a preferred embodiment, the quaternary amine solution is selected from ammonium chloride solution, ammonium hydroxide solution and mixtures thereof.

According to the invention, the term "quaternary amine solution" means a solution in which the quaternary form of the amine represents at least 51% of the forms of the amine.

In a preferred embodiment, the quaternary amine solution contains only the quaternary amine in aqueous solution. According to another preferred embodiment, the quaternary amine solution at a basic pH, preferably between 10 and 13, more preferably 11. The results presented in Example 3 demonstrate the advantages of quaternary amine solutions with regard to conventional solutions of cell lysis. Surprisingly, the quaternary amine solution has in fact the best cell lysis and assay levels of the molecules of the ECM.

During this lysis step, the quaternary amine solution is brought into contact with the cells in culture, in a state of confluence and preferably after complete or partial withdrawal of the culture medium.

The quaternary amine solution is applied at a concentration sufficient to lyse the cells. Studies conducted by the Applicant have shown that a sufficient concentration for lysing the cells is generally in a range from 100 μΜ to 2M. According to a preferred embodiment, the concentration of the quaternary amine solution is from 1 mM to 200 mM, more preferably from 10 mM to 100 mM, still more preferably 20 mM. In the particular case where the culture medium has not been removed or only partially, the dilution factor is considered to adjust the concentration of the quaternary amine solution.

These studies, part of which are presented in Examples 2, show in particular that the preferential lysis time is between 5 and 60 minutes. Indeed, the results obtained with longer times are not better and do not justify longer lysis times.

According to an advantageous embodiment, the quaternary amine solution is chosen from an ammonium chloride solution at a concentration ranging from 100 μΜ to 2M, preferably from 1 mM to 200 mM, preferably from 10 mM to 100 mM, preferentially to a concentration of 20 mM, and among a solution of ammonium hydroxide at a concentration ranging from 100 μΜ to 2M, preferably from 1 mM to 200 mM, preferably 10 mM to 100 mM, preferably at a concentration of 20 mM and their mixtures . According to an alternative method, the quaternary amine solution is preferably chosen by comparison with the results obtained with the 20 mM ammonium hydroxide solution for 10 minutes and gives, in particular, when tested under the same conditions, the same molecule ratio. MEC / DNA at plus or minus 16%, and preferably without significant difference with ammonium hydroxide at 20mM for 10 minutes.

According to a preferred embodiment, the concentration and the application time of the quaternary amine solution, preferably ammonium, are chosen in comparison with the results obtained with a lysis solution of 20 mM ammonium hydroxide applied during 10 minutes. Thus, the ratio of the result of the determination of the molecule of the MEK obtained in step e) on the result of the assay of the DNA in the cell lysate is compared with that obtained with a solution of lysis of ammonium hydroxide with 20mM applied during the same time.

Preferably, the comparison is carried out by a One Way Anova variances statistical analysis test and the concentration and the application time are chosen so that the ratio of the tested solution is not significantly different for at least p. <0.05 and preferably p <0.001.

The lysis step is preferably carried out at ambient temperature, preferably between 15 ° C. and 25 ° C., preferably 20 ° C. It is preferably carried out with stirring.

The method according to the invention then comprises a step of sampling the cell lysate.

According to an advantageous embodiment, the cell lysate is analyzed.

According to a preferred embodiment, a step of measuring the amount of DNA is performed in order to rationalize the assay results of the ECM molecules by amount of DNA. This embodiment thus makes it possible to compare the results reported indirectly with the number of viable cells in culture. In the case of an evaluation of an ingredient of cosmetic interest

And / or pharmaceutical in particular, this embodiment thus allows to express the effect observed on the molecule of the ECM, for example stimulation by cell.

The step of measuring the amount of DNA in the lysate can be carried out according to the usual techniques in the field for example a biochemical assay using intercalating agent revealed by fluorescence.

According to an advantageous mode, the molecule of the ECM studied and contained in the cell lysate, optionally in a precursor form, is assayed by immunological method, preferably by ELISA type assay.

The method according to the present invention then comprises a step of immunoassay of the molecule contained in the extracellular matrix, preferably by the fluorescence method TRF.

In a preferred embodiment, the ECM molecule assay results are streamlined by DNA level. The fluorescence measurement can be converted to the amount of ECM molecule through a standard range in the usual manner in this type of assay.

According to a preferred embodiment, the culture medium taken in step b) and / or the cell lysate taken in step d) are analyzed to advantageously allow the MEC molecule to be assayed in the different compartments: intracellular, in the culture medium and in the MEC.

The present invention also relates to a method for in vitro assay by immunological technique of at least one molecule of the extracellular matrix synthesized by cells in culture comprising at least the successive steps:

a) a step of culturing the cells

B) a step of collecting the culture medium and assaying the ECM molecule and / or a precursor of the ECM molecule

c) a step of lysing the cells with a quaternary amine solution

d) a step of collecting the cell lysate and assaying the DNA and / or assaying the molecule of the MEK and / or a precursor of the molecule of the MEK e) a step of immunological assay of said molecule of the ECM contained in the extracellular matrix.

The preferred embodiments have been described previously. This method advantageously makes it possible to assay the molecule of the ECM in the various compartments: intracellularly, in the culture medium and in the MEK.

The present invention also relates to the use of the assay method according to the invention for the screening of one or more ingredients of cosmetic and / or pharmaceutical interest.

Thus the subject of the present invention is also a method for screening and / or studying in vitro at least one ingredient of cosmetic and / or pharmaceutical interest for its properties to increase or decrease the quantity of a molecule of the matrix. extracellular. The screening and / or study method according to the invention comprises the assay method according to the invention described above in which the cells are cultured in the presence of the ingredient. During step a) the culture of confluent cells in the culture medium containing the ingredient of cosmetic and / or pharmaceutical interest is carried out for at least 24 hours and preferably from 24 to 96 hours, preferably for 48 hours. .

Advantageously, the screening and / or study method according to the invention comprises at least the successive steps:

a) a step of culturing the cells and culturing the confluent cells in the presence of the ingredient

b) preferentially a step of sampling the culture medium and even more preferably of assaying the molecule of the MEK and / or a precursor of the molecule of the MEK in said culture medium thus taken,

c) a step of lysing the cells with a quaternary amine solution d) a step of sampling the cell lysate and preferably of assaying the DNA and / or assaying the molecule of the MEK and / or a precursor of the molecule of the MEK

e) a step of immunologically assaying said MEC molecule contained in the extracellular matrix.

In a preferred embodiment, the method further comprises a step f) of determining the properties of the ingredient to increase or decrease the amount of a molecule of the extracellular matrix.

Determination of the ability to stimulate or inhibit the ingredient is then performed with respect to an experiment conducted without ingredient and / or conducted with a so-called positive control ingredient. In the case where collagen is the molecule of the MEK assayed, the positive control is preferably selected from Vitamin C at a concentration which ranges from 5 μΜ to 100 μΜ and preferentially to 50 μΜ, the TGF beta preferentially at a dose that ranges from 1 ng. / ml at 100ng / ml, more preferably at 10ng / ml and mixtures thereof. Ascorbic acid and TGF beta are indeed so-called collagen "booster" ingredients and the studies carried out by the Applicant have demonstrated their interest in the assay method according to the invention (Example 5).

The ingredient is then classified according to the ratio of the MEK molecule assay to the e) stage rationalized on the DNA measured in step d) compared to the ratio of the assay performed without ingredient and / or with a positive control optimized on the DNA measured in step d). The comparison is made preferably by a statistical analysis test of variances said One Way Anova.

If the ratio obtained of the ingredient is significantly greater than that obtained with the untreated evidence for at least p <0.05 and preferably p <0.001, then the ingredient increases the functionality of the molecule in the MEC c. ie its content in the MEC if the ratio of the ingredient is significantly greater than that obtained with ascorbic acid and / or TGF beta for at least p <0.05 and preferentially p <0.001, alo rs the ingre die nt increases the functionality of the collagen in the MEC ie its content in

5 the MEC

if the ratio of the ingredient is significantly lower than that of the untreated control for at least p <0.05 and preferentially p <0.001, then the ingredient decreases the functionality of the molecule in the MEC, that is to say its content in the MEC

An ingredient of cosmetic interest having the capacity to increase the content of MEK molecules assayed in the MEK, in particular for collagen in a manner greater than 50 μΜ ascorbic acid, may be used for the cosmetic treatment, in particular particularly for the anti-aging treatment, in particular for reducing wrinkles and the signs of skin aging, and / or for improving the firmness

15 of the skin.

An ingredient of pharmaceutical interest having the capacity to increase the content of MEK molecules assayed in the MEK, in particular for collagen in a manner greater than vitamin C at 50μΜ can be used to improve the healing process, for the treatment of osteoarthritis.

An ingredient of pharmaceutical interest having the ability to decrease the molecule content of ECM can be used for the treatment of fibrosis and hypertrophic scars.

Another subject of the present invention is an immunological assay kit useful for the implementation of the assay method according to the invention and / or for the method for screening and / or studying an ingredient of the invention. cosmetic and / or pharmaceutical interest according to the invention comprising a quaternary amine solution and an antibody directed against the ECM molecule. The preferred and / or advantageous characteristics of the quaternary amine solution have previously been described in the context of the method according to the invention.

The antibody directed against the molecule of the ECM is a monoclonal or polyclonal antibody and preferably selected from an anti-collagen antibody, an anti-elastin antibody and mixtures thereof.

The antibody can be measured, optionally by coupling with a secondary antibody by enzyme-substrate type EIA (Enzyme Immuno Assay) assays, in particular ELISA (Enzyme-Linked Immunosorbent Assay), radiolabelling (RIA) and radioimmunoassay. by fluorescence measurement said FIA (Fluorescent immunoassay) in particular by time-resolved fluorescence or so-called TRF (Time Resolved fluorescence).

According to a preferred embodiment, the assay kit according to the invention further comprises a solution of ascorbic acid at a preferential concentration ranging from 5μΜ to 100μΜ and preferably to 50μΜ.

According to a preferred embodiment, the assay kit according to the invention also comprises an antibody directed against the precursor of the ECM molecule and / or a bisbenzimide solution for the determination of the DNA in the method according to the invention.

The present invention also relates to the use of a dosing kit according to the invention for carrying out the method according to the invention.

The present invention also relates to a method for selecting a lysis solution that is useful in the assay method according to the invention, characterized in that the assay method according to the invention is implemented with a lysis solution with test for at least 10 min, preferably 10 min, and the ratio of the result of the determination of the molecule of the MEC obtained to

Step e) on the result of the assay of the DNA in the cell lysate is compared with that obtained with a lysis solution of ammonium hydroxide at 20 mM applied during the same time, preferably 10 minutes.

Preferably, the comparison is carried out by a statistical analysis test of the One Way Anova variances and the lysis solution tested is selected if the ratio of the test solution is not significantly different for at least p <0.05 and preferably p <0.001.

The lysis solution thus selected can be used in place of the quaternary amine solution in the assay method according to the invention. The present invention thus also relates to the lysis solutions thus selected according to the selection method according to the invention.

According to a preferred embodiment, the methods according to the invention consist only of steps a), b), c), d) e) and optionally f).

Other objects, features and advantages of the invention will become apparent to those skilled in the art upon reading the explanatory description which refers to examples which are given by way of illustration only and which do not in no way limit the scope of the invention.

The examples are an integral part of the present invention and any features appearing novel from any previous state of the art from the description as a whole, including the examples, form an integral part of the invention in its function and function. in its generality.

Thus, each example has a general scope.

On the other hand, in the examples, and unless otherwise indicated, the temperature is expressed in degrees Celsius, and the pressure is atmospheric pressure unless otherwise indicated.

EXAMPLES

EXAMPLE 1 Assay Method for Type I Collagens in the ECM 25 According to the Invention

Principle:

The molecule of the ECM assayed by the method according to the invention is the type I collagen synthesized by fibroblasts.

Protocol:

Step a): Normal human fibroblasts obtained from abdominal biopsies are seeded in a 96-well plate and cultured in a defined medium (FGM) to 100% confluence which was obtained after 3 days of culture.

- Step b):

After 48 hours of post-confluence culture, the culture medium is removed and removed.

- Step c):

A lysis step is carried out: 1 10 μl of a solution of ammonium hydroxide at 20 mM for 10 minutes at room temperature and with stirring, step d)

The lysate is removed and analyzed. The double-stranded DNA contained in the lysate is assayed by bisbenzimide method (Picogreen Invitrogen Kit).

- Step e)

The assay of collagen in the MEK is carried out directly on the MEK by an anticollagen antibody I. A secondary antibody coupled to europium was used with a revealing solution and the fluorescence was measured, (Deifia® kit - Perkin Elmer ).

The fluorescence results were rationalized with respect to the measured dsDNA level.

The experiment was carried out on 6 wells (n = 6)

The results presented are the mean (Avg) and the standard deviation (EC). Results:

25 Discussions: Since the amount of fluorescence is proportional to the amount of collagen measured, the amount of collagen can be determined using a standard range.

EXAMPLE 2 Study of the Influence of the Lysis Time on the Type I Collagen Assay Method in the MEC According to the Invention

Principle:

The molecule of the ECM assayed by the method according to the invention is the type I collagen synthesized by the fibroblasts as a function of the lysis time.

o Protocol:

The protocol described in Example 1 is applied with different durations of the lysis step with the 20mM ammonium hydroxide solution ranging from 5 minutes to 240 minutes.

The experiment is carried out for each duration on 6 wells (n = 6)

The results presented correspond to the average (Avg) and the standard deviation (EC) and the significance determined by One way Anova analysis (Dunnett) with regard to the optimum duration of 10 minutes.

NS: not significant NT: not analysable

^* : significant at p <0.05 ^** : significant at p <0.01 ^*** : significant at p <0.001 20 Results:

Table 2

Conclusion:

It is observed that at 2.5 minutes the extracted DNA is too small to be indicative of the amount of viable cells. From 5 minutes, the amount of collagen measured and the amount of dsDNA are homogeneous over time. The collagen is not degraded by the ammonium hydroxide solution over time and the optimal lysis time is determined at 10 minutes. Experiments conducted during lysis step times of up to 48 hours were performed and confirmed these observations.

EXAMPLE 3 Study of the Influence of the Nature of the Lysis Solution on the Type V Collagen Assay Method in the MEC According to the Invention

Principle:

The MEC molecule assayed by the method according to the invention is the type V collagen synthesized by fibroblasts and the study is conducted according to the nature of the lysis solution.

Protocol:

The protocol described in Example 1 is applied in the same way. Thus normal human fibroblasts obtained from abdominal biopsies are seeded in 96 well plate and cultured in a defined medium (FGM) up to 100% confluence.

After 48 hours of culture after confluence at 37 ° C. under 0.5% CO2, the culture medium is removed.

The lysis solutions tested are as follows:

solution according to the invention NH40H: 1 10 μl of a solution of ammonium hydroxide at 20 mM.

solution 1: solution containing 52.55 g of urea, 15.01 g of thiourea, 964 mg of DTT and 37.2 mg EDTA

solution 2: solution containing Tris 10mM / Triton 0.1%

- solution 3: solution containing Hepes 50mM

solution 4: solution containing 50 Mm Tris HCl pH7.5), detergent agent (0.5mM deoxycholate sodium) and a chelating agent (20mM EGTA)

solution 5: solution containing EDTA 50mM

solution 6: solution containing 0.2N NaOH and 1% SDS

The application time of the lysis solution tested is 10 minutes at room temperature and with stirring.

The collagen V assay in the ECM is carried out directly on the ECM by an anti-collagen V antibody. A secondary antibody coupled to europium was used with a revealing solution and the fluorescence was measured, (Déifia® kit - Perkin Elmer ).

Fluorescence is measured.

The experiment is performed for each lysis solution on 6 wells (n = 6)

The results are shown in Table 3.1

The experiment is renewed and the lysis solutions tested are as follows: solution according to the invention NH 4 OH: 1 10 μl of an ammonium hydroxide solution at concentrations of between 200 μΜ and 2M.

- Monoethylamine solution at 20mM

- 20mM diethylamine solution

5 - Triethylamine solution at 20 mM

The results are presented in Table 3.2 and 3.3

The results presented correspond to the mean (Avg) and the standard deviation (EC) and the significance determined by One way analysis Anova (Dunnett) with regard to the NH4OH lysis solution.

î o NS: not significant NT: not analysable

^* : significant at p <0.05 ^** : significant at p <0.01 ^*** : significant at p <0.001 Results:

Table 3.1:

Table 3.2:

Table 3.3:

NH40H solution

200 μM 2 mM 20 mM 200 mM 2 M Coll V Medium 114177 117380 104228 118318 1 19292

(RFU) EC 5889 6187 4757 6504 2839

DNA Avg 282.65 259.39 303.01 397.80 512.48

EC 21, 05 25.23 10.98 41, 95 34.27

Ratio: Avg 403.95 452.53 343.98 297.43 232.77

Coll / EC 41, 81 77.75 12.79 41, 73 17.50

DNA% 117.43 131, 56 100.00 86.47 67.67

Anova ns ns - ns ns

(Dunnett)

Discussions:

The tests carried out show that the ammonium hydroxide solution makes it possible to obtain more reliable, repeatable and reproducible results than the conventional cell lysis solutions and the primary, secondary and tertiary amine solutions. The ammonium hydroxide solution allows assays under maximum conditions for both cell lysis and collagen V measurement. The other quaternary amine solutions tested have also shown very interesting results in both cell lysis. and the collagen V measurement. Furthermore, the collagen V / DNA ratios obtained with different concentrations of ammonium hydroxide lysis solution are not significantly different from that obtained with a 20 μm solution. This concentration solution is optimal because of its practicality of use.

EXAMPLE 4: Type I collagen assay method in the three compartments according to the invention

Principle:

The different compartments: intracellular (cell lysate), culture medium and ECM are recovered and the different forms of collagen I are assayed.

20

Protocol:

The method described in Example 1 is applied and the culture and lysis media removed are analyzed.

The experiment is carried out without active ingredient on the one hand and with ascorbic acid solubilized in PBS at different concentrations of 0.1 to 100 μΜ. On the culture medium taken, an assay of the amount of procollagen is carried out (kit PIPc, TAKARA,) by ELISA to determine the amount of extracellular procollagen (Table 4.1)

On the lysing medium taken, an assay of the amount of procollagen (PIPc kit, TAKARA,) by ELISA is performed to determine the amount of intracellular procollagen (Table 4.2). An assay of the amount of DNA (Picogreen kit, invitrogen) is also performed to rationalize the set of measurements in each well to the amount of viable cells.

The dosage of the amount of collagen I in the MEK is carried out and presented in table 4.3

The amounts of collagen and procollagen measured are related to the amount of DNA present in each well.

A One Way Anova statistical analysis was performed against the witness. NS: not significant NT: not analysable

15 ^* : significant at p <0.05 ^** : significant at p <0.01 ^*** : significant at p <0.001

Results:

Table 4.1:

Table 4.2

Active Ingredient | witness | Ascorbic acid (μΜ)

Table 4.3:

Control active ingredient Ascorbic acid (μΜ)

0.1 1 10 50 100

Coll 1 Avg 75204 74156 74419 78694 79146 79796

ADN Moy 340 318 303 281 279 291

Ratio: Avg 221, 83 233.65 246.46 280.82 284.31 273.96 Coll 1 /

DNA

Stat Moy 100.00 105.33 1 1 1, 10 126.59 128.17 123.50

EC 3.92 3.61 6.19 6.82 7.86 4.72

Dunnett - ns ** ** ** **

Discussions:

The amount of procollagen and collagen can be calculated by reference to a standard curve of procollagen and human collagen, respectively.

Ascorbic acid or vitamin C is known to increase the synthesis and secretion of procollagen in the extracellular medium which is also demonstrated. These stimulatory effects are also measured with the method according to the invention, including at the level of the collagen I content in the MEK. This effect is dose-dependent up to 50 μΜ where the threshold effect of the molecule is observed. The concentration observed in the intracellular medium under the effect of vitamin C is lower than that observed with the untreated control since procollagen was secreted in the extracellular medium more importantly.

EXAMPLE 5 Use of the Type I and Type V Collagen Assay Method as a Screening Method: Principle:

Active ingredients of cosmetic interest known for their stimulating effect of collagen synthesis are tested for their property on type I collagen in ECM and then on type V collagen in ECM.

5 Protocol:

The experiment is carried out according to the protocol described in Example 1 from fibroblasts extracted from a biopsy of a donor aged 63 years.

The active ingredients of cosmetic interest that have been tested are:

- Ascorbic acid 50 μΜ solubilized in a PBS buffer solution is tested at different concentrations of 5 to 100 μΜ.

a soy protein hydrolyzate described in the patent application WO2009121422 and marketed by the Applicant under the name Phytokine ™ is tested at different concentrations of 0.5 to 2% v / v

The confluent cells were incubated in the presence of the active ingredients for 48 hours at 37 ° C. under 0.5% CO ₂ or without active ingredient (control) for 48 hours at 37 ° C. under 0.5% CO _2.

The experiment is performed for each condition on 6 wells (n = 6) - The result presented is the mean (Avg) and the standard deviation (EC)

The results obtained for the determination of type I collagen under the effect of Phytokine ™ are shown in Table 5.1 and those for the determination of type V collagen in Table 5.2.

The results obtained for the determination of type I collagen under the effect of ascorbic acid are shown in Table 5.3 and those for the determination of type V collagen in Table 5.4.

Significance is calculated by One Way Anova (Dunnett) test against the control.

NS: not significant NT: not analysable

Table 5.1: Active Ingredient Phytokine ™ Control (% v / v)

0.5 1 1.25 1.5 2

Coll 1 Avg 78857 86149 81474 84256 82888 85938

(RFU) EC 793 1496 1806 3592 2868 2093

ADN Avg 282.67 284.89 271.07 272.17 260.96 254.26

EC 6.8 7.0 4.8 7.6 6.9 8.0

Ratio: Avg 277.54 302.06 300.42 307.51 315.94 341 .07

Coll / DNA EC 10.4 1.8 16.2 25.3 19.4 15.6

Versus Avg 100 108.84 108.25 1 10.80 1 12.98 122.89

Witness EC 2.62 4.09 3.28 6.00 4.23 5.22

Dunnett - * * * * *

Table 5.2:

Active Ingredient Phytokine ™ Control (% v / v)

0.5 1 1.25 1.5 2

Coll V Mm 13479 13483 141 13 14451 15452 16818

(RFU) EC 560 1096 870 593 751 716

ADN Avg 268.68 259.81 250.56 250.72 247.90 222.37

EC 7.9 5.2 7.7 7.7 5.5 8.6

Ratio: Avg 49.9 51 .86 56.29 57.80 62.38 72.52

Coll / ADN EC 1 .4 5.3 1.5 3.0 2.5 6.7

Versus Avg 100 103.92 1 12.80 1 15.84 125.01 151 .34

Witness EC 6.3 9.84 10.68 7.72 8.75 10.69

Dunnett - NS NS * * *

Table 5.3:

Active Ingredient Control Ascorbic acid (μΜ)

5 10 25 50 100

Coll. 1 Av 5251 1 59802 59569 59088 54454 50765

(RFU) EC 3160.78 3278.42 2991, 90 1412.56 5177.36 5084.09

ADN Moy 242 220 249 232 180 204

EC 17.34 23.70 9.28 13.62 30.42 25.35

Ratio: Avg 217.51 273.40 239.53 254.84 309.87 249.31

Coll / DNA EC 12.52 14.74 7.88 13.00 66.93 9.50

Versus Avg 100.00 125.69 1 10.12 1 17.16 130.66 1 14.62

Witness EC 5.36 7.29 3.70 6.44 10.30 4.09

Dunnett - * * * * *

Table 5.4:

Active Ingredient Control Ascorbic acid (μΜ) 5 10 25 50 100

Coll V Avg 12337 12644 14127 16172 16230 17994 (RFU) EC 1 103.62 734.56 1072.20 971, 01 1 125.26 460.87

DNA Moy 241 212 241 236 236 246

EC 1 1, 90 12.22 8.57 3.51 6.75 5.67

Ratio: Avg 51, 07 59.83 58.76 68.69 68.86 73.12 Coll / DNA EC 2.40 4.56 6.39 5.29 5.32 2.83

Versus Mean 100.00 1 17.16 1 15.06 134.49 134.82 143.17 Indicator EC 6.10 8.17 1 1, 46 9.63 9.32 5.08

Dunnett - * * * * *

Discussions:

The results show that Phytokine ™ induces a synthesis stimulation of collagen I and V with a dose effect and significantly as does ascorbic acid. For the determination of collagen I the optimum dose of efficacy of ascorbic acid is 50 μΜ.

EXAMPLE 6 Use of the Type I Collagen Assay Method as a Screening Method

Principle:

Active ingredients of cosmetic interest known for their stimulating effect on collagen synthesis are tested for their property on type I collagen in ECM.

Protocol:

The active ingredients of cosmetic interest that have been tested are:

50 μΜ ascorbic acid solubilized in a PBS buffer solution

- vegetal extract tested at 1% (v / v) which is an aqueous extract of Davilla rugosa leaves

The confluent cells were incubated in the presence of the active ingredients for 48 hours at 37 ° C. under 0.5% CO 2 or without active ingredient (control) for 48 hours at 37 ° C. under 0.5% CO _2. The experiment is performed for each condition on 6 wells (n = 6) - The result presented is the mean (Avg) and the standard deviation (EC)

The significance is calculated by one-way test Anova (Dunnett)

NS: not significant NT: not analysable

*: significant at p <0.05 ^** : significant at p <0.01 ^*** : significant at p <0.001

Results:

Discussions:

The results show that the ascorbic acid at 50μΜ and the tested plant extract induce an increase in the amount of collagen I in the MEK significantly. The tested plant extract is more effective than ascorbic acid and is therefore an ingredient of cosmetic and / or pharmaceutical interest that can be used in particular for the cosmetic treatment of wrinkles and for increasing cutaneous firmness.

EXAMPLE 7 Study of the properties of ingredients screened with the method according to the invention

- dosage of procollagen in the culture medium:

In experiment 6 procollagen was assayed in the culture medium taken in step b) according to the method described in Example 4.

The results are shown in Table 7.

Witness EC 14.98 31.53 3.5

Dunnett - *** NS

Conclusion: The plant extract does not show an increase in procollagen synthesis in the culture medium but has demonstrated an increase in the amount of collagen I in the matrix.

5 - immunohistochemical analysis

Protocol: An immunohistochemical analysis was performed according to the following protocol:

Normal human fibroblasts from a biopsy were seeded on 8 well labteck slides at 40,000 cells / cm 2 and cultured in FGM defined culture medium to 100% confluency. The evaluated ingredients, plant extract 1% v / v or ascorbic acid 50μΜ were applied for 72h. After 72 hours of post-confluence culture, the culture medium is removed and immunostaining is performed. The polyclonal anti-collagen I primary antibody is applied and after removal of the medium the secondary antibody coupled to a

15 fluorochrome alexa 488 is added.

Observation of the fluorescence is performed using the LSM700 confocal microscope.

Results: The results measured according to the fluorescence are as follows:

Conclusion:

The visualization study made it possible to confirm the results obtained according to the invention. These results also demonstrate the interest of screener an ingredient according to its properties on the quantity of molecules in the ECM and justify the interest of the method according to the invention.

Claims

1. In vitro immunological technique assay method for at least one extracellular matrix molecule synthesized by cells in culture comprising at least the steps of:

a) a step of culturing the cells, and preferentially culturing the confluent cells for at least 24 hours,

b) a preferential step of sampling the culture medium,

c) a step of lysing the cells with a quaternary amine solution,

d) a step of collecting the cell lysate,

2. Assay method according to claim 1 wherein step b of harvesting the culture medium is carried out.

3. assay method according to one of the preceding claim wherein the molecule of the ECM is selected from the constitutive proteins of ECM, glycoproteins constitutive of EC EC, glycoaminoglycans constitutive of the ECM, the proteoglycans constitutive of the ECM and the

20 growth factors contained in the MEC.

4. Assay method according to claim 3 wherein the molecule of the ECM is selected from collagens type I, III, V, VI, XII, XIV, XVI, IV and VII, elastin, tropoelastin, fibrillin 1, LOX, LOXL, ΙΈΒΡ (Elastin Binding Protein), fibullins 3 and 5, Emilin 1 and 2, Perlecan, Versican,

Decorin, biglycan, lumican, fibronectin, laminin, hyaluronic acid, heparan sulfate.

The assay method of claim 3 wherein the ECM molecule is selected from VEGF, PDGF, FGF-2, FGF-7, and HGF.

6. assay method according to one of the preceding claims wherein the cells in culture are selected from are stromal cells, preferably fibroblasts, osteoblasts and / or adipoblasts, epithelial, preferably keratinocytes or endothelial cells.

The assay method of claim 6 wherein the cells in culture are selected from fibroblasts and / or keratinocytes and / or adipocytes.

8. Assay method according to one of the preceding claims wherein the cells in culture are maintained in culture at the confluence stage for a period of time which goes from 24 to 96 hours before the completion of step c).

9. Assay method according to claim 8 wherein an active ingredient of cosmetic and / or pharmaceutical interest is added to the culture medium.

10. Assay method according to claim 9 wherein the ingredient is selected from vitamin C, preferably at a dose ranging from 5μΜ to 100μΜ and TG F beta preferentially at a dose ranging from 1 ng / ml to 100ng / ml .

11. Assay method according to one of claims 2 to 10 wherein the molecule of the ECM and / or a precursor of the ECM molecule is determined by immunological method in the culture medium taken in step b) preferably by assay type ELISA.

12. Assay method according to one of the preceding claims wherein the am uternary am ulation solution is an ammonium solution,

Optionally in the form of salts.

13. Assay method according to the preceding claim wherein the quaternary amine solution is selected from an ammonium chloride solution, an ammonium hydroxide solution and mixtures thereof.

14. Assay method according to one of the preceding claims wherein the quaternary amine solution is at a concentration ranging from 10mM to

200mm.

15. Assay method according to one of the preceding claims wherein the quaternary amine solution is applied to the cells for a time ranging from 5 to 60 minutes.

16. Assay method according to one of the preceding claims wherein a DNA and / or molecule assay of the ECM is carried out in the cell lysate taken in step d).

17. Assay method according to one of the preceding claims wherein the step e) immunological assay of said molecule

Contained in the extracellular matrix is performed by TRF fluorescence method.

18. Assay method according to one of the preceding claims comprising at least the steps:

b) a step of collecting the culture medium and assaying the ECM molecule and / or a precursor of the ECM molecule,

c) a step of lysing the cells with a quaternary amine solution; d) a step of collecting the cell lysate and assaying the DNA and / or assaying the ECM molecule and / or a precursor of the molecule of the ECM e) a step of immunological assay of said molecule of the ECM contained in the extracellular matrix.

19. Use of the assay method according to one of the preceding claims for the screening and / or the in vitro study of at least one ingredient of cosmetic and / or pharmaceutical interest for its properties to increase or decrease the amount of a molecule in the extracellular matrix.

20. A method for screening and / or in vitro study of at least one ingredient of cosmetic and / or pharmaceutical interest for its properties to increase or decrease the quantity of a molecule in the extracellular matrix comprising at least the steps :

a) a step of culturing the cells,

b) preferably a step of sampling the culture medium and still more preferably of assaying the molecule of the MEK and / or a precursor of the molecule of the MEK in said culture medium thus collected,

c) a step of lysing the cells with a quaternary amine solution; d) a step of collecting the cell lysate and preferentially assaying the DNA and / or assaying the MEC molecule and / or a precursor of the molecule of the MEC,

e) a step of immunological assay of said molecule of the ECM contained in the extracellular matrix.

21. An immunological assay kit useful in accordance with one of the preceding claims comprising a quaternary amine solution and an antibody directed against the ECM molecule, optionally coupled to a secondary antibody.

22. A method of selecting a lysis solution useful in the assay method according to one of claims 1 to 18 or according to the screening method.

25 according to claim 20, characterized in that the assay method according to the invention is carried out with a lysis solution to be tested for at least 10 min, preferably 10 min, and the ratio of the result of the assay of the molecule of the ECM obtained in step e) on the result of the determination of the DNA in the cell lysate in step d) is compared with that obtained with a solution of lysis of ammonium hydroxide at 20 mM applied during the same time, preferably 10 minutes.